Goals: The long-term goal of our project is to shed light on the molecular mechanism of protein biosynthesis. Ribosomes are the universal cell organelles facilitating the translation of the genetic code into proteins. These nucleoprotein assemblies (mw 2.3 mega-Dalton, about 4500 RNA nucleotides and 75 proteins) are built of two subunits of unequal size (0.85 and 1.45 mega-Dalton), which associate upon the initiation of protein biosynthesis. The immediate objectives of this proposal are (a) to use the high-resolution structures of the two eubacterial ribosomal subunits determined by us as references for the elucidation of the structures of ribosomal complexes with substrate analogs, functional ligands, inhibitors and antibiotics; (b) to exploit these structures for illuminating the mechanisms involved in peptide bond formation, decoding, translocation, inhibition by and resistance to antibiotics; (c) to progress toward high resolution structure determination of complexes capturing the whole ribosome at defined functional states; (d) to advance towards the determination of the structure of eukaryotic ribosomes. Methods: Highly active ribosomes from robust organisms are being crystallized. X-ray diffraction data are being collected at cryogenic temperatures from flash-frozen crystals, using state-of-the-art high brilliance synchrotron radiation. Phases are being determined by a combination of MIRAS, molecular replacement and crystal averaging, and used to locate the bound ligands and antibiotics in difference electron density maps. The significance of ribosomal crystallography stems from its potential to illuminate the mechanism of a fundamental life process, protein biosynthesis. The already known structures revealed the modes of action of several antibiotic agents that target the ribosome, illuminated principles of drug selectivity, and provided significant basic knowledge of possible pathways of drug resistance. These, and further to come insights, should lead to improved therapeutic agents and allow structure based design of a new generation of more powerful, selective and efficient antibiotics.